Effect of neutrophil extracellular traps on tumor lymph nodes.

Abstract

4033 Background: In most human cancers, regional lymph nodes (LNs) are the first sites of metastasis. In addition to being an important part of the tumor staging system, with the advent of novel therapies, lymph node metastasis has become a crucial clinical intervention point before distant metastasis, the leading cause of cancer-associated deaths. To initiate metastasis, the conditions of LNs need to be optimized for tumor cell deposition and growth. This process is believed to be mediated by the activation of immune cells including polymorphonuclear neutrophils (PMNs). However, the cellular mechanism is not well defined. Our early observations suggest that PMNs and neutrophil extracellular traps (NETs), DNA comprising structures that are extruded in response to inflammatory cues, are associated with adverse oncologic outcomes. Thus, one potential mechanism of increased LN metastasis is that tEVs recruit PMNs and propend NETs formation. Methods: Human tissue micro-arrays (TMAs) of gastroesophageal (GEA) cancer patients were stained with PMN and NETs markers and quantified by HALO software. C57BL/6 or pad4-/- mice were injected with B16F10 or H59 cells alone or treated with neutrophil elastase inhibitor (NEi) or PMN depletion antibody. LN sections were stained with NETs markers and quantified by ImageJ (NIH). Results: In the study of 175 GEA cancer patients, lymphatic NET deposition was observed in both tumor infiltrated lymph nodes and tumor negative lymph nodes. We also demonstrated high LN NETs deposition was associated with reduced survival, even in the absence of overt metastasis ( p=0.03). Next, we sought to investigate the dynamic and the consequence of LN NETs deposition using animal models. We found that LN Neutrophil Recruitment and NET deposition happens in a pre-metastatic manner. Moreover, LN metastasis was abrogated through different kinds of NETs inhibition (neutrophil depletion, pad4 knockout and NEi treatment, n=10, p<0.001), demonstrating the consequences of LN NETs deposition and its potential as a treatment target. Finally, we showed that the LN PMN recruitment and NETs formation was mediated by increased production of IL-8 by Lymphatic Endothelial Cells (LEC). Conclusions: Together, we demonstrated that NETs can contribute to LN metastasis, and can serve as a potential therapeutic targets. By further investigating the detailed mechanism, this project will lead to major advances in the management of cancer patients.